The realization of the remarkable potential of human embryonic stem cells or induced pluripotent stem cells to treat human disease cannot occur unless new animal-free methods for the expansion of undifferentiated stem cells are developed. An optimal extracellular matrix (ECM) substitute for the expansion of human embryonic stem cells (hESCs) in three dimensions (3-D) would be a soft hydrogel that includes two key features. First, the hydrogel should be composed of animal-free components to which researcher-provided ECM proteins or synthetic peptides can be covalently attached. Second, after multiple hESC divisions in an undifferentiated state, the hydrogel should allow rapid recovery of the expanded cell numbers without altering stemness. Glycosan BioSystems currently markets HyStem", an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan ("Glycosil") for culture of a variety of pluripotent cell types. In Phase I we will test the feasibility of a simple system for attachment of proteins to the hydrogels that would covalently bind any selected human matricellular protein to the thiol-modified HA component of HyStem. This approach directly couples the "pull" of the cells on the proteins to the elastic modulus of the gel. In our first Aim, we would establish the optimal combination of covalently-linked human collagen, vitronectin, fibronectin, and laminin (CVFL) proteins, as well as the minimum amounts required, for effective self-renewal of H9 hESCs. Cells will be recovered and analyzed for stem cell markers by flow cytometry. Second, for each combination that gave suitable cell growth, we will employ a new reductively-cleavable linker to form soft gels that will allow enzyme-free recovery of undifferentiated hESCs from the gel surface. After cell recovery with 25 mM NAc-Cys, stemness will be assessed as before. Third, H9 hESCs will be encapsulated in 3D in selected HyStem-CVFL hydrogels, expanded in 3-D, and analyzed. Finally, we will use optimized conditions to expand stem cells for a minimum of 6 passages, and compare the karyotypes and stem cell markers with cells cultured on murine fibroblast feeders, on Matrigel, or on CVFL- coated plastic. PUBLIC HEALTH RELEVANCE: The realization of the remarkable potential of human embryonic stem cells or induced pluripotent stem cells to treat human disease cannot occur unless new animal-free methods for the expansion of undifferentiated stem cells are developed. Glycosan BioSystems currently markets HyStem", an in situ-crosslinkable, semi- synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil") for culture of a variety of pluripotent cell types. The goal of this proposal is to tailor HyStem for human embryonic stem cell culture by determining the optimal combination of covalently-bound attachment factors for proliferation without differentiation and by including a fall-apart crosslinker for gentle cell recovery.